M. tuberculosis is a natural mutant in the putative central regulator of oxidative stress response, oxyR. All M. tuberculosis strains investigated to date have nearly identical lesions in oxyR, and the elimination of oxyR function may have coincided with or directly participated in the evolution of M. tuberculosis into the potent contemporary human pathogen. The loss of oxyR has a dual significance for M. tuberculosis pathogenesis and treatment of tuberculosis. First, this surprising phenomenon reflects the complexity of host-parasite interactions and its continuing analyses are expected to reveal strategies used by M. tuberculosis for survival in the host. Second, the loss of oxyR and the associated dysfunction of oxidative stress responses most likely contribute to the exceptional sensitivity of M. tuberculosis to isonicotinic acid hydrazide (INH). The goals of this proposal are: (i) to delineate the effects of the loss of oxyR on M. tuberculosis response to reactive oxygen and nitrogen intermediates. This will be accomplished by identifying genes and functions affected by the loss of oxyR, analyzing alternative modes of their regulation, and characterizing M. tuberculosis response to reactive oxygen and nitrogen; (ii) to determine whether and how oxyR inactivation and the resulting dysfunction of oxidative stress responses contribute to the exceptional sensitivity of M. tuberculosis to INH. This will be examined by complementation of the defect in M. tuberculosis using a functional mycobacterial oxyR, by inactivation of oxyR in model mycobacteria, and by determination of the effects that such modifications have on INH sensitivity in vitro and in vivo; (iii) to investigate the role of the loss of oxyR in M. tuberculosis virulence and pathogenesis using mouse bone marrow derived macrophages and human monocytes, and models of tuberculosis in mice and guinea pigs. In addition to conventional analyses of M. tuberculosis survival and histopathology, GFP-based technologies developed in the PI's laboratory will be employed to monitor mycobacterial localization, and potential tissue- and infection stage-specific expression of oxidative stress response genes (e.g. ahpC). These experiments will delineate the effects that the loss of oxyR has on host-pathogen interactions in tuberculosis. The proposed studies are expected to improve understanding of the elusive virulence attributes of M. tuberculosis, and may help explain, at least in part, its high sensitivity to the front-line antituberculosis agent INH.